CN108508200A - Detect the method and its application of the cell of CD19 CAR - Google Patents

Detect the method and its application of the cell of CD19 CAR Download PDF

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Publication number
CN108508200A
CN108508200A CN201810350458.8A CN201810350458A CN108508200A CN 108508200 A CN108508200 A CN 108508200A CN 201810350458 A CN201810350458 A CN 201810350458A CN 108508200 A CN108508200 A CN 108508200A
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Prior art keywords
antibody
car
detection
cd19car
detection reagent
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CN108508200B (en
Inventor
答亮
王宏飞
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Shanghai Xingwan Biotechnology Co ltd
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Shanghai Shang Luo Bio Pharmaceutical Technology Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Abstract

The present invention provides a kind of method and its application of detection CD19 CAR positive cells.Specifically, the present invention provides a kind of detection reagent for detecting CD19 CAR positive cells, the detection reagent includes：(a) the first detection reagent, first detection reagent is first antibody, and the first antibody is polyclonal antibody, and the polyclonal antibody specifically binds to the extracellular antigen binding domain of CD19 CAR；(b) the second detection reagent, second detection reagent specifically bind to first antibody or are coupled to the marker of first antibody.The detection reagent and method high sensitivity of the present invention, specific good, testing result more accuracy.

Description

Detect the method and its application of the cell of CD19 CAR
Technical field
The invention belongs to biotechnologys and medical domain, and in particular to a method of the cell of detection expression CD19 CAR And its application.
Background technology
With the development of immunotherapy of tumors, the Chimeric antigen receptor of antibody and immunocyte advantage is combined (chimeric antigen receptor, CAR)-T cell immunization therapy is of great interest.
CAR is mainly made up of cell membrane exoantigen combined area and intracellular signal transduction area hinge area and transmembrane region. Film exoantigen combined area has the function of specific recognition and combines target cell surface antigen, constitutes derived from monoclonal antibody Single-stranded Variable domain (Single Chain Variable Fragment, scFv), by heavy chain variable region (VH) and light chain Variable region (VL) forms.Intracellular signal transduction area is mainly by the CD3zeta chain groups of costimulatory signal and T cell receptor (TCR) At.After the T cell (CAR-T) of expression CAR is combined by scFv with target cell antigen, signal is passed to T by intracellular signal transduction area Cell plays lethal effect to activate T cell, secretion perforin, granzyme and cell factor etc..
FDA approvals at present have listed two CAR-T cell drugs, are the Kymriah of Novartis (Novartis) respectively, With the Yescarta of lucky Leadd B.V (Gi lead).Two drug is directed to CD19 positive B-cells tumours, uses identical Antigen binding domain derives from the scFv of mouse monoclonal antibody FMC63.
Structure CAR-T cells need to transfect CAR genes by virus or non-viral system and are integrated into T cell genome On.When the gene normal expression, when forming the CAR structures of cross-film on cell membrane, CAR-T cells, which just have, identifies and kills target The activity of cell.Therefore, the positive T cell of accurate detection expression CAR is the committed step of CAR-T Control of drug quality, and Clinically to the control of Case treatment dosage, process monitoring and with the important link diagnosed.
Common type there are two ways to being detected for CAR-T cells：Detect CAR gene masculines T cell and inspection Survey CAR protein positive T cells.
For detecting CAR gene masculine T cells, quantitative real time aggregation integrated enzyme reaction (Quantitative Real Time PCR, qPCR) it is the main method for detecting CAR gene masculine T cells.It can be detected by qPCR whole in cellular genome The CAR genes of conjunction and the copy number of CAR genes.But this method cannot accurately reflect the positive T cell of expression CAR, because having The T cell that a part incorporates CAR genes does not have normal expression CAR (Jennifer N.Brudno, et al., Journal of Clinical Oncology, 2016), the false positive that these T cells are brought reduces the accuracy of qPCR methods application.In addition, QPCR methods cannot be satisfied the demand to a variety of surface markers of T cell (CD3, CD4, CD8 etc.) while detection, this is further Limit the application of qPCR methods.
For detecting CAR protein positive T cells, existing detection reagent includes anti-mouse IgG (Fab ')2(Jackson ImmunoResearch companies), Protein L (Zhi li Zheng, et al., Journal of Translational Medicine,2012)、CD19/Fc(Satiro N De Oliveira,et al.,Journal of Translational Medicine, 2013), GFP-CD19 (number of patent application 201610354642.0), mouse monoclonal antibody (Pub.No.: US20170342164A1, clone 136.20.1) etc..However, these existing detection reagents still have the shortcomings that, detection Effect is unsatisfactory.Wherein, anti-mouse IgG (Fab ')2It is poor with the specificity of Protein L detection CD19 CAR, it can not Distinguish the different target spot CAR (such as CD22CAR, CD123CAR etc.) of mouse source difference scFv structures.CD19/Fc and GFP-CD19 are equal Dependent on the combination of the parts scFv on CD19 albumen and CD19 CAR, since the affinity of the scFv and CD19 is substantially less than complete Whole antibody (FMC63) and CD19 affinity (IAN C.Nicholson, et al., Molecular Immunology, 1997), therefore the sensitivity of CD19/Fc and GFP-CD19 detection CD19 CAR is relatively low.Mouse monoclonal antibody (clone number 136.20.1 CD19 CAR) are detected, combination of the monoclonal antibody to scFv on CD19 CAR are depended on, since the two is same kind (mouse source), affinity is relatively low, therefore there are still the not high defects of sensitivity for monoclonal antibody detection.
Therefore, there is an urgent need in the art to develop to detect sensitive CAR positive cells detection reagent and inspection high, accuracy is high Survey method, to meet the needs of CAR-T Control of drug quality, clinical treatment and adjoint diagnosis.
Invention content
It is an object of the invention to provide a kind of sensitive CAR positive cells detection reagent high, accuracy is high of detection and its Using.
In the first aspect of the present invention, a kind of detection reagent for detecting CD19 CAR positive cells is provided, it is described Detection reagent includes：
(a) the first detection reagent, first detection reagent is first antibody, and the first antibody is polyclonal antibody, And the polyclonal antibody specifically binds to the extracellular antigen binding domain of CD19 CAR；With
(b) the second detection reagent, second detection reagent specifically bind to first antibody or are coupled to first antibody Marker；
And detection sensitivity >=5 positive cell/1000 the CD19 CAR cell of the detection reagent.
In another preferred example, the first antibody is unmodified polyclonal antibody.
In another preferred example, second detection reagent is the secondary antibody that specificity is directed to the first antibody.
In another preferred example, the first antibody is biotinylated polyclonal antibody, and described second is examined Test agent is the Avidin avidin or Streptavidin streptavidin of biotin specific bond.
In another preferred example, detection sensitivity >=2 positive cell/1000 CD19 CAR of the detection reagent are thin Born of the same parents, more preferably >=1 positive cell/1000 CD19 CAR cell.
In another preferred example, the polyclonal antibody is rabbit source polyclonal antibody.
In another preferred example, the first antibody and secondary antibody are from different mammalian species.
In another preferred example, the first antibody is rabbit source, and secondary antibody is donkey source or Yang Yuan.
In another preferred example, the CAR positive cells include CAR immunocytes and the nonimmune cells of CAR.
In another preferred example, the CAR positive cells are selected from the group：T cell, NK cells, CIK cell, NKT are thin Born of the same parents, or combinations thereof.
In another preferred example, the CAR positive cells include：HEK-293T cells, Jurkat cell or its group It closes.
In another preferred example, the polyclonal antibody also has one or more of feature：
(i) and the potency of the combination of CD19 CAR scFv recombinant proteins is 1:1000-1:20000, preferably 1：2000- 1：10000；
(ii) it is 10 with the affinity dissociation constant of CD19 CAR scFv recombinant proteins-10-10-12M,
In another preferred example, it the polyclonal antibody specific recognition and is incorporated into based on anti-CD19 monoclonal antibodies The CD19 CAR of the scFv structures of FMC63.
In another preferred example, the extracellular antigen binding domain of the CD19 CAR has SEQ ID No.:Shown in 1 Amino acid sequence：
DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISN LEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDY GVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDY WGQGTSVTVSS(SEQ ID No.:1)
In another preferred example, the CD19 CAR scFv recombinant proteins have CD19 CAR scFv-His tag knots Structure.
In another preferred example, the CD19 CAR scFv recombinant proteins have SEQ ID No.:Amino described in 2 Acid sequence：
DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISN LEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDY GVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDY WGQGTSVTVSSHHHHHH(SEQ ID No.:2)。
In another preferred example, the polyclonal antibody is prepared by the following method：
(a) CD19 CAR scFv recombinant proteins are provided；With
(b) rabbit is immunized with the recombinant protein, to obtain the anti-blood for the CD19 CAR scFv recombinant proteins (i.e. polyclonal antibody) clearly.
In another preferred example, the CD19 CAR scFv recombinant proteins have SEQ ID No.:Amino described in 2 Acid sequence：
In another preferred example, the CD19 CAR scFv recombinant proteins include the recombination of eukaryotic expression or prokaryotic expression Albumen.
In another preferred example, the CD19 CAR scFv recombinant proteins are the recombinant proteins of Bacillus coli expression.
In another preferred example, the CD19 CAR scFv recombinant proteins are by renaturation process.
In another preferred example, the CD19 CAR scFv recombinant proteins are eukaryotic expressions.
In another preferred example, the CD19 CAR scFv recombinant proteins are to be prepared by the following method acquisition：
(s1) expression plasmid is provided, the expression plasmid contains the expression cassette of CD19 CAR scFv recombinant proteins；
(s2) eukaryotic host cell is transfected with the expression plasmid, to obtain the eukaryotic host cell through transfection；
(s3) under conditions suitable for the expression, the eukaryotic host cell through transfection is cultivated, to express the recombination Albumen；With
(s4) recombinant protein is detached from the cultivating system.
In another preferred example, the expression includes secretion type expression (being secreted into cell culture fluid).
In another preferred example, the eukaryotic host cell is selected from the group：HEK-293T cells, Chinese hamster ovary celI, COS-1 Cell, COS-7 cells.
In the second aspect of the present invention, a kind of method preparing first antibody is provided, the first antibody is polyclonal Antibody, and the polyclonal antibody specifically binds to the extracellular antigen binding domain of CD19 CAR；
It the described method comprises the following steps：
(a) CD19 CAR scFv recombinant proteins are provided；With
(b) rabbit is immunized with the recombinant protein, to obtain the anti-blood for the CD19 CAR scFv recombinant proteins (i.e. polyclonal antibody) clearly.
In another preferred example, the CD19 CAR scFv recombinant proteins have SEQ ID No.:Ammonia shown in 1 or 2 Base acid sequence.
In another preferred example, the method further includes optional step
(c) polyclonal antibody obtained in (b) is detached and/or is purified；With
(d) optionally to described, the polyclonal antibody through detaching and/or purifying carries out performance measurement.
In another preferred example, the performance measurement includes one or more detections selected from the group below：
(i) specific detection；
(ii) bioactivity；
(iii) sensitivity technique.
In the third aspect of the present invention, a kind of CD19 CAR detection kits, the detection kit are provided：
(t1) the first container, and the first antibody in the first container, the first antibody are Anti-TNF-α Body, and the polyclonal antibody specifically binds to the extracellular antigen binding domain of CD19 CAR；With
(t2) second container, and resist for the second of the polyclonal antibody positioned at the specificity of the second container Body；And
(t0) specification.
In another preferred example, the secondary antibody is the antibody of anti-rabbit IgG.
In another preferred example, the secondary antibody is selected from the group：The antibody of donkey anti-rabbit IgG, goat anti-rabbit igg it is anti- Body.
In another preferred example, the secondary antibody carries detectable label.
In another preferred example, the detectable label includes fluorescence.
In another preferred example, the secondary antibody is the secondary antibody of fluorescent marker.
In another preferred example, the detection kit further includes containing one or more additional detections selected from the group below Reagent：
(t3) third container, and the sealer in the third container；
(t4) the 4th container, and positive control (such as CD19 CAR scFv recombination eggs in the 4th container In vain)；With
(t5) the 5th container, and the negative control reagent in the 5th container.
In another preferred example, the additional detection reagent is located in identical or different container.
In another preferred example, the method that the specification describes detection CD19 CAR positive cells.
In the fourth aspect of the present invention, a kind of purposes of detection reagent as described in the first aspect of the invention is provided, is used In the reagent or kit that prepare detection CD19 CAR positive cells.
In another preferred example, the CAR positive cells include CAR immunocytes and the nonimmune cells of CAR.
In another preferred example, the CAR positive cells are selected from the group：T cell, NK cells, CIK cell, NKT are thin Born of the same parents, or combinations thereof.
In another preferred example, the CAR positive cells include：HEK-293T cells, Jurkat cell or its group It closes.
In the fifth aspect of the present invention, a kind of method of detection CD19 CAR positive cells, including step are provided：
(I) detection reagent described in claim 1 is provided；
(II) cell mass to be detected is detected with the detection reagent, to obtain CD19 CAR positive cells Qualitative or quantitative testing result.
In another preferred example, the method includes step：
(1) 0.5 × 10 is taken5-5×105Cell to be measured, with buffer solution (such as PBS contains 1% sealer) washing containing sealer 1-3 times；
(2) buffer solution (such as PBSs, containing 1% closing of the 50-200 μ l containing sealer and first antibody (polyclonal antibody) is used Polyclonal antibody such as R19mcar described in agent and 0.5-2%) it is resuspended cell, and (preferably placed at 4 ± 2 DEG C at 2-15 DEG C A period of time T1 (such as 20-90 minutes, be preferably about 30-60min)；
(3) cell is washed 1-3 times with buffer solution (such as PBS)；
(4) using buffer solutions of the 50-200 μ l containing the secondary antibody with detectable marker (such as fluorophor), (PBS contains 0.5-2% secondary antibodies) be resuspended cell, and 2-15 DEG C (preferably place a period of time T2 at 4 ± 2 DEG C (and such as 10-60 minutes, preferably Ground about 20-40min)
(5) cell is washed 1-3 times with buffer solution (such as PBS)；
(6) cell is resuspended with a certain amount of (such as 100-500 μ l) buffer solution (such as PBS), and machine carries out on flow cytometer Detection.
In another preferred example, the method is external detection method.
In another preferred example, the method is nondiagnostic and non-therapeutic method.
In another preferred example, the method is quality control method.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment) It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.
Description of the drawings
Fig. 1 shows the result of rabbit anti-serum detection CD19 CAR-T cells of the present invention.
Fig. 2 shows the bioactivity result of rabbit anti-serum of the present invention.
Fig. 3 shows the testing result for using rabbit anti-serum R19mcar of the present invention and other reagents to CD19 CAR-T cells Compare.
Fig. 4 show with rabbit anti-serum R19mcar of the present invention detections CD19 CAR-T's as a result, wherein with existing mouse it is single The testing result of anti-136.20.1 compares.A- detects CAR-T cells using the mouse monoclonal antibody 136.20.1 of control；B- is originally The R19mcar of invention detects CAR-T cells；C- mouse monoclonal antibodies detect CAR-T cells；The R19mcar of the D- present invention detects CAR-T Cell.
Fig. 5 is shown detects CD19 CAR-T's as a result, wherein with CD19/Fc's with rabbit anti-serum R19mcar of the present invention Data and mouse monoclonal antibody 136.20.1 data compare.
Fig. 6 shows the results contrast being detected to CD19 CAR-T and CD22CAR-T using different reagents.
Fig. 7 shows the result of Immunofluorescence test CD19 CAR positive cells.
Specific implementation mode
The present inventor after extensive and in-depth study, by largely screening, unexpectedly develops a kind of to CAR-T for the first time The detection sensitivity of cell is high, accuracy is good and specific good detection reagent.The present invention detection reagent be based on optimization, The rabbit prepared with the specific correctly recombinant antigen of configuration is mostly anti-.The experimental results showed that based on detection reagent of the present invention Detection method, not only can be directly against the extracellular antigen binding domain on CAR-T cells, but also detection sensitivity is existing far above at present All kinds of detection reagents having, in addition, differentiation of the detection reagent of the present invention for positive CAR-T cells and feminine gender CAR-T cells Spend very high, testing result is more accurate.The present invention is completed on this basis.
Term
As used herein, term " first antibody of the present invention ", " polyclonal antibody of the present invention ", " present invention is mostly anti-", " this hair Bright antiserum ", " rabbit anti-serum of the present invention " etc. are used interchangeably, and referring to using the present invention optimized has specific correct structure The recombinant antigen of type, carrying out immune and preparation the rabbit anti-serum specifically bound with the recombinant antigen to rabbit, (i.e. rabbit is more It is anti-).In the present invention, the recombinant antigen corresponds to the extracellular antigen binding domain in CAR structures.For example, when being directed to CD19 When CAR-T cells are detected, a kind of typical recombinant antigen is FMC63scFv.
As used herein, term " polyclonal antibody " refers to the composition for including different antibody molecules, these antibody point The a variety of different specific epitopes or react that son can be attached on identical or different antigen.
As used herein, term " CAR " refers to Chimeric antigen receptor, including extracellular domain, transmembrane domain and cell Intracellular domain.Extracellular domain includes target-specific binding members (also referred to as antigen-binding domains).Intracellular domain packet Include costimulatory signal conducting region and ζ chain parts.Costimulatory signal conducting region refers to the intracellular domain including costimulatory molecules A part.Costimulatory molecules are lymphocyte to the required cell surface molecule of the effective response of antigen.
As used herein, " single chain variable fragment (scFv) " refers to the single chain polypeptide from antibody, remains combination The ability of antigen.The example of scFv includes the antibody polypeptides formed by recombinant DNA technology, wherein heavy chain immunoglobulin (H Chain) it is connected via intervening sequence with the areas Fv of light chain (L chains) segment.
The present invention is mostly anti-
One important feature of the detection reagent of the present invention is first antibody, and the first antibody is polyclonal antibody, and And the polyclonal antibody specifically binds to the extracellular antigen binding domain of CD19 CAR.
When (secondary antibody is that specificity is directed to described first to first antibody of the invention with corresponding secondary antibody Antibody) combination when, not only can specifically be detected, but also to may be up to >=1 CD19 CAR positive thin for detection sensitivity Born of the same parents/1000 cell.
In another preferred example, the polyclonal antibody is rabbit source polyclonal antibody.
In another preferred example, the first antibody and secondary antibody are from different mammalian species.
In another preferred example, the first antibody is rabbit source, and secondary antibody is donkey source or Yang Yuan.
In another preferred example, the polyclonal antibody also has one or more of feature：
(i) and the potency of the combination of CD19 CAR scFv recombinant proteins is 1:1000-1:20000, preferably 1：2000- 1：10000；
(ii) it is 10 with the affinity dissociation constant of CD19 CAR scFv recombinant proteins-10-10-12M,
Kit
The present invention also provides one kind for detecting CD19 CAR detection kits, the detection kit：
(t1) the first container, and the first antibody in the first container, the first antibody are Anti-TNF-α Body, and the polyclonal antibody specifically binds to the extracellular antigen binding domain of CD19 CAR；With
(t2) second container, and resist for the second of the polyclonal antibody positioned at the specificity of the second container Body；And
(t0) specification.
In another preferred example, the secondary antibody is the antibody of anti-rabbit IgG.
In another preferred example, the secondary antibody is selected from the group：The antibody of donkey anti-rabbit IgG, goat anti-rabbit igg it is anti- Body.
In another preferred example, the secondary antibody carries detectable label.
In another preferred example, the detectable label includes fluorescence.
In another preferred example, the secondary antibody is the secondary antibody of fluorescent marker.
In another preferred example, the detection kit further includes containing one or more additional detections selected from the group below Reagent：
(t3) third container, and the sealer in the third container；
(t4) the 4th container, and positive control (such as CD19 CAR scFv recombination eggs in the 4th container In vain)；With
(t5) the 5th container, and the negative control reagent in the 5th container.
In another preferred example, the additional detection reagent is located in identical or different container.
In another preferred example, the method that the specification describes detection CD19 CAR positive cells.
Detection method
The present invention also provides one kind to be resisted more based on the present invention, the method being detected to CAR positive cells.Due to this hair It is bright how anti-for matching or there are high-affinity and high specific in the antigen binding domain of corresponding CAR, therefore the present invention's is more It is anti-to can be used for efficient, sensitive and accurately detect CAR positive cells.
Include (but being not limited to) for the CAR positive cells of the method for the present invention detection, representative example can be used：T is thin A variety of different cells such as born of the same parents, NK cells, NKT cells, CIK cell.For example, being directed to the rabbit polyclonal of FMC63scFv with the present invention For antibody (R19mcar), can be used for the detections of corresponding CD19 CAR positive cells, (including T, NK, NKT or CIK are thin Born of the same parents).
The detection method of the present invention, can be used for scientific research, cell drug research and development, cell drug quality control, cell drug faces Bed Treatment monitoring, clinical patient are with diagnosis etc..
Main advantages of the present invention include：
(a) (dissociation constant Kd is 10 for the affinity of rabbit antibody of the present invention-10-10-12M is horizontal) relative to mouse monoclonal antibody, (Kd exists 10-9-10-10M is horizontal), 10-1000 times, therefore the detection reagent of the CAR positive cells based on the how anti-exploitation of the present invention can be improved Box, detection sensitivity is high, specificity is good.
(b) detection reagent and method of the invention are very high for the discrimination of CAR positive cells and CAR negative cells, Testing result more accuracy.
(c) the more anti-scFv for mouse source of the present invention have higher affinity (being significantly better than mouse source monoclonal antibody).
(d) the how anti-detection reagent of rabbit of the invention compares anti-mouse IgG (Fab ')2、Protein L、CD19/Fc、GFP- The existing detection reagent such as CD19, mouse monoclonal antibody, can it is more special, sensitiveer, more efficiently detect CD19 CAR positive cells.
(e) mostly anti-and kit the production cost of rabbit of the present invention significantly reduces, therefore is applied to CAR-T drug quality controls System and clinical treatment and adjoint diagnosis are advantageously.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, no Then percentage and number are weight percent and parts by weight.
Universal method
The activation and CD19 CAR slow-virus infections of 1.T cells
(1) 0.5x10 is taken6T cell (0.5 × 106/ ml), it is placed in 24 orifice plates；
(2) by specification guidance washes the magnetic bead for being coated with AntiCD3 McAb/CD28 3 times with R10 culture solutions；
(3) magnetic bead is pressed 3：1 (magnetic bead：Cell) ratio addition T cell, place 37 DEG C, 5%C02It was cultivated in incubator Night；
(4) after t cell activation 12-24 hours, 24 orifice plates is centrifuged, 800 μ l supernatant culture solutions are removed；
(5) melt lentiviral particle at room temperature, gently mixing, 1ml lentiviral particles are added 0.5 × 106T cell In (200 μ l), polybrene to 8 μ g/ml of final concentration is added, sets in centrifuge 2500 turns, 1.5 hours, after centrifugation, puts Set 37 DEG C, overnight incubation in 5%C02 incubators；
(6) 24 orifice plates of centrifuged overnight culture remove most of supernatant culture solution containing lentiviral particle, are added fresh R10 culture solutions expand T cell；
(7) it counts T cell within every 2 days, IL-2 50IU/ml is added, maintain T cell in 0.5-1 × 106/ml；
(8) after 3-5 days, 2 × 10 are taken5Cell passes through flow cytomery CD19 CAR-T cells.
2. flow cytomery CD19 CAR-T cells
(1) 2 × 10 are taken5The T cell of CD19 CAR slow-virus infections is washed 2 times with PBS, and cell is resuspended with 100 μ l PBS；
(2) the detection antibody (or other detection reagents) of corresponding dilution ratio is added, 4 DEG C, places 45min；
(3) PBS is washed 2 times；
(4) addition fluorescence secondary antibody, 4 DEG C, avoid light place 30min；
(5) PBS is washed 2 times；
(6) 300 μ l PBS are resuspended, machine testing on flow cytometer.
Embodiment 1.
The preparation method of FMC63scFv rabbit polyclonal antibodies (R19mcar)
1. the preparation and purification of recombinant antigen
Build the scFv-His tag recombinant antigens of CAR containing CD19 sequence (amino acid sequence such as SEQ ID No.:Shown in 2) Expression plasmid, transfect HEK-293T cells.Recombinant antigen is expressed through HEK-293T cells and is secreted into cell culture fluid, into And utilize this albumen of Ni affinity chromatographys column purification.
(1) by HEK-293T cells adhere-wall culture in 10cm culture dishes, 37 DEG C, 5%CO2Overnight incubation.
(2) according to 2000 specifications of Lipofectamine, CD19 CAR scFv-His tag recombinant antigen sequences will be contained Expression plasmid, transfect HEK-293T cells.
(3) cell is removed in the 6th day harvest culture solution, centrifugation after transfecting, and obtains supernatant.
(4) according to QIAGEN companies Ni-NTA Superflow specifications, the antigen protein in supernatant is purified.
The preparation resisted 2. more
Use the recombinant protein of purifying as antigen, immune rabbit, method is as follows：
(1) by the Fo Shi Freund's complete adjuvants of 0.5ml and 0.5ml antigens (200 μ g) mix well.
4 positions (back and thigh root), each position 250ul is immunized in (2) 1 rabbits.
(3) it is 20 days that the period, which is immunized, and blood examination is taken within 7-10 days to survey antiserum titre after being immunized, and is immunized 4-5 times in total.
The purifying resisted 3. more
Rabbit is isolated and purified by proteinA from rabbit anteserum to resist more.Method is as follows：
(1) sample preparation：Supernatant is taken after 3ml rabbit anti-serums are centrifuged, 4 times of volumetric balance buffer solution (20mM are used in combination Na2HPO4, 0.15M NaCl, pH 7.0) and it is diluted processing；
(2) column operation is filled：It fully shakes up so that resin (Protein-A media) is resuspended, draws 3ml slurries to new chromatographic column In, it is previously added 3ml equilibration buffers in chromatographic column, allows resin natural subsidence, outflow equilibration buffer that it is slow that 10ml balances are added Resin is balanced in fliud flushing to chromatographic column, flows out equilibration buffer by the flow velocity of about 1ml/min；
(3) chromatographic purifying：By sample according in the flow velocity loading to chromatographic column of about 1ml/min, efflux is collected, after being used for The continuous binding ability for measuring resin.With the equilibration buffer solution resin of 20ml, flow velocity maintains about 2ml/min.It is eluted with 10ml Buffer solution (0.1M glycine, pH 3.0) antibody elution, flow velocity maintain about 1ml/min, collect containing purposeful immunoglobulin Eluent, and it is added immediately the neutralization buffer (1M Tris, pH 8.5) of 1/10 elution buffer volume, adjust pH to 7.4. It is detected with spectrophotometer after being concentrated into 1-1.5ml with super filter tube.
As a result：
(1) after purified, a concentration of 0.1-3.0mg/ml of recombinant antigen
(2) in 4 immunized rabbits, there are 3 antiserums for only producing high-titer, take the highest antiserum of potency It (is named as：R19mcar) it is used for subsequent experimental.
(3) after purified, antibody concentration 0.1-2.0mg/ml
Embodiment 2.
Antiserum detects CD19 CAR-T cells
In the present embodiment, the rabbit polyclonal antibody prepared using embodiment 1, by Flow cytometry, to CD19 CAR-T cells are detected.
In flow cytometer detection, detection antibody is antiserum (dilution ratio is as shown in Figure 1), and fluorescence secondary antibody is donkey anti-rabbit secondary antibody (488 fluorescent markers of Alexa Fluor).
The result shows that rabbit (4) and mouse (6) is immunized with same antigen, completes after being immunized, animal blood serum is taken to be examined It surveys.In 4 immune rabbits, 3 successfully generate specific antibody, and rabbit anteserum presses 1：1000 dilutions use, and can normally detect CD19 CAR positive T cells.
For immune result with rabbit on the contrary, 6 immune mouse are all without generating specific antibody, mouse serum can not detect CD19 CAR positive T cells.
The above results illustrate, for recombinant antigen prepared in embodiment 1.1, can efficiently be generated in rabbit special Property be directed to CD19 CAR antibody.
In addition, the highest antiserum of potency is taken (to be named as：R19mcar) it is used for subsequent experimental.
Embodiment 3.
Rabbit anti-serum bioactivity
Flow cytometer detection method is with embodiment 2, wherein the gradient dilution liquid (thinner ratio that detection antibody is antiserum R19mcar Example is shown in Fig. 2), fluorescence secondary antibody is donkey anti-rabbit secondary antibody (488 fluorescent markers of Alexa Fluor).
The results are shown in Figure 2.The 1 of the positive rabbit anti-serum of the present invention：100 to 1：5000 dilutions can be detected normally CD19 CAR positive T cells illustrate potency up at least >=1：5000.
Embodiment 4.
Detection of the different detection reagents to CD19 CAR-T cells
In the present embodiment, using different detections, CD19 CAR-T cells are detected.The CD19CAR-T cells Group contains CAR positive T cells.
Flow cytometer detection method is with embodiment 2, wherein the detection antibody (reagent) used is respectively the antiserum of the present invention R19mcar、Rabbit anti mouse(Fab’)2(488 fluorescent markers of Alexa Fluor), Goat anti mouse (Fab’)2(488 fluorescent markers of Alexa Fluor), GFP-CD19, Protein L (biotin labels).R19mcar is corresponded to Fluorescence secondary antibody be donkey anti-rabbit secondary antibody (488 fluorescent markers of Alexa Fluor), the corresponding reagents of Protein L (biotin labels) For FITC-Streptavidin.
The results are shown in Figure 3, wherein
(a) when antiserum R19car using the present invention, CD19 CAR-T positive cells and negative cells divide group it is apparent ( In fluidic cell figure, positive cell group is irised out with dotted line).
(b) in contrast, other reagents can not clearly distinguish positive and negative cells.
Therefore, for detection accuracy and signal strength, R19mcar is far superior to Rabbit anti mouse (Fab’)2、Goat anti mouse(Fab’)2, GFP-CD19, the detection reagents such as Protein L.
There is high accuracy since the present invention is sero-fast, and can obviously distinguish the CAR positives and negative cells, therefore can The accurate geo-statistic CD19 CAR positive T cells to facilitate, this is particularly significant for drug Quality Control, clinical treatment.
Embodiment 5.
R19mcar detects CD19 CAR-T cells
Flow cytometer detection method is with embodiment 2, wherein detection antibody is R19mcar, fluorescence secondary antibody is donkey anti-rabbit secondary antibody (488 fluorescent markers of Alexa Fluor).Compared with mouse monoclonal antibody 136.20.1, R19mcar detect CD19 CAR-T the positive and Negative cells divide group apparent.
As shown in Figure 4 A, when detecting CAR-T cells using the mouse monoclonal antibody 136.20.1 of control, positive signal and negative signal It partly overlaps (frame).
As shown in Figure 4 B, when detecting CAR-T cells with the R19mcar of the present invention, positive signal is complete with negative signal It separates (frame).
It as shown in Figure 4 C,, all can not be with no matter CD4 is positive or the CD8 positive CAR-T when mouse monoclonal antibody detection CAR-T cells Negative T cell is completely separable (frame).
As shown in Figure 4 D, when detecting CAR-T cells with the R19mcar of the present invention, positive signal is complete with negative signal It separates (frame).
Therefore, for detection accuracy and signal strength, how anti-R19mcar of the invention is far superior to mouse monoclonal antibody 136.20.1。
In addition, cooperation specificity is directed to the detection method of CD4 and/or CD8, the method for the present invention further can be distinguished effectively Two subgroups of CD4 positive T cells and CD8 positive T cells of the CAR positives.
Embodiment 6.
R19mcar detection sensitivities compared with other reagents are analyzed
In the present embodiment, the CD19 CAR-T cells for being used for detection are diluted with negative T cell by corresponding proportion.Streaming is examined Survey method is with embodiment 2, wherein detection antibody is R19mcar, fluorescence secondary antibody is that (Alexa Fluor 488 are glimmering for donkey anti-rabbit secondary antibody Signal).
The results are shown in Figure 5.When R19mcar detects dilution ratio to 1/1024, CD19 CAR positive T cell percentages For 0.15% (being 57.0% when original undiluted).
When mouse monoclonal antibody 136.20.1 detects dilution ratio to 1/1000, CD19 CAR positive T cell percentages are 0.300% (being 87.2% when original undiluted), for the two under the conditions of 1/1000 dilution ratio, sensitivity is suitable.
Document report is in addition, there will be, CD19/Fc (number is AF488-CD19sIg1-4 in text) can detect 0.5% CAR-T positive cells, corresponding highest dilution ratio are 1/128.
Therefore, for detection sensitivity, R19mcar is better than mouse monoclonal antibody 136.20.1, more far superior to CD19/Fc.
Embodiment 7.
R19mcar detects specificity compared with Protein L
Flow cytometer detection method is with embodiment 2, wherein detection antibody (reagent) is respectively R19mcar and Protein L (biotin labels).The corresponding fluorescence secondary antibodies of R19mcar are donkey anti-rabbit secondary antibody (488 fluorescent markers of Alexa Fluor), The corresponding reagents of Protein L (biotin labels) are FITC-Streptavidin.
It is clinical at present to grind a variety of CAR-T including targeting CD22, and carry out the continuous filling treatments of a variety of CAR-T and double targets It is developed to CAR-T.It is particularly significant for medicament research and development, Quality Control, clinical treatment accurately to detect and distinguish different CAR-T.
As shown in fig. 6, R19mcar can be with specific detection CD19 CAR-T, nonrecognition CD22CAR-T (solid box)；And Protein L identify two kinds of CAR-T, and detection signal is similar (dotted line frame), detect without specificity.Therefore, in detection specificity, R19mcar is better than Protein L, and detection reagent (anti-mouse similar with Protein L mechanism, without CAR-T target spot specificity IgG(Fab’)2)。
Embodiment 8.
R19mcar detects CD19 CAR-T cells
In the present embodiment, the rabbit polyclonal antibody prepared using embodiment 1, by immunofluorescence technique, to CD19 CAR- T cell is detected.Method is as follows：
(1) cell is fixed with 4% formaldehyde 20 minutes, method is formaldehyde to be directly added into culture medium and is adjusted carefully at room temperature Born of the same parents are to 1 × 106/ml；
(2) 1mL cell solutions are added in 1.5ml microcentrifugal tubes, are centrifuged 30 seconds in micro centrifuge；
(3) it pours out supernatant and cell precipitation is resuspended in 1ml PBS, supernatant is poured out in centrifugation, and by cell precipitation It is resuspended in 200 μ l PBS；
(4) 5 μ L cell suspensions are added to the glass slide for being coated with gelatin, pipette tip is used in combination to smear；
(5) slide is placed on hot plate (low-heat), and liquid is made slowly to evaporate, surround using barrier pen hydrophobic barrier Cell blots simultaneously air-dry；
(6) washing contains the glass slide of fixed cell twice in 400 μ L washing buffers (PBS containing 0.1%BSA)；
(7) 400 μ L Block buffers (PBS for containing 10% donkey serum) are added and block unspecific staining, incubate at room temperature It educates 35 minutes, discards Block buffer；
(8) 1 is pressed in dilution buffer (PBS containing 1%BSA, 1% donkey serum)：100 dilution R19mcar antibody, in room Temperature is lower to be incubated 1 hour；
(9) it is washed twice in 400 μ L washing buffers；
(10) according to 1 in dilution buffer：1000 dilution Alexa488 goat antirabbits Ig (H+L) secondary antibodies, at room temperature It is protected from light incubation 1 hour；
(11) it is rinsed twice with 400 μ L washing buffers；
(12) the diluted DAPI solution of 300 μ L (1 is added per hole：1000) it, and is incubated at room temperature 2-5 minutes；
(13) it is rinsed once, is rinsed with water primary with PBS；
(14) moisture is carefully absorbed, 1 drop is prevented that mountant, which is quenched, to be dropped on microscopic slide, and use appropriate size Coverslip；
(15) fluorescence microscope.
With the CD19 CAR slow-virus infection Jurkat cells without GFP labels, CD19 CAR-Jurkat are obtained.It is immune Fluorescence results (left figure, CD19 CAR-Jurkat as shown in Figure 7；Right figure, Jurkat), R19mcar can clearly dye positive thin Born of the same parents, green represents CD19 CAR normal presentations on cell membrane on film.The Jurkat for being uninfected by CD19 CAR slow virus is thin Born of the same parents' then redgreen fluorescence signal.
Embodiment 9
A kind of CD19 CAR positive cell flow cytometer detection kits using R19mcar
A kit is prepared, it includes following reagent：
(a) the first container and the R19mcar in the first container；
(b) second container and in the second container fluorescence secondary antibody (wherein, fluorescence can be selected FITC, PE, Fluorescence different Alexa Fluor 488 etc.)；
(c) third container and the NC (negative control reagent) in the third container；With
(d) the 4th container and the sealer in the third container.
The kit application method is as follows：
(1) 2 × 10 are taken5Cell to be measured is washed 2 times with PBS (containing 1% sealer)；
(2) cell is resuspended (containing 1% sealer, 1%R19mcar) with 100 μ l PBS, 4 DEG C, places 45min；
(3) PBS is washed 2 times；
(4) it is resuspended cell with 100 μ l PBS (contain 1% fluorescence secondary antibody), 4 DEG C, avoid light place 30min；
(5) PBS is washed 2 times；
(6) 300 μ l PBS are resuspended, machine testing on flow cytometer.
CD19 CAR-T cells are detected with the kit, the results showed that, which has high sensitivity, spy Anisotropic good effect.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To be made various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.
Sequence table
<110>Shanghai Shang Luo biological medicines Science and Technology Ltd.
<120>Detect the method and its application of the cell of CD19 CAR
<130> P2018-0539
<160> 2
<170> PatentIn version 3.5
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<213>Artificial sequence（Artificial sequence）
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Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
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Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
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Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
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Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
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Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr
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Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu
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Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys
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Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg
145 150 155 160
Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser
165 170 175
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Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly
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Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val
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Ser Ser
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35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
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Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
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Ser Ser His His His His His His
245

Claims (10)

1. a kind of for detecting the detection reagents of CD19CAR positive cells, which is characterized in that the detection reagent includes：
(a) the first detection reagent, first detection reagent is first antibody, and the first antibody is polyclonal antibody, and The polyclonal antibody specifically binds to the extracellular antigen binding domain of CD19CAR；With
(b) the second detection reagent, second detection reagent specifically bind to first antibody or are coupled to the mark of first antibody Remember object；
And detection sensitivity >=5 positive cell/1000 the CD19CAR cell of the detection reagent.
2. detection reagent as described in claim 1, the polyclonal antibody is rabbit source polyclonal antibody.
3. the extracellular antigen binding domain of detection reagent as described in claim 1, the CD19CAR has SEQ ID No.:Amino acid sequence shown in 1：
DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSL TISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVS LPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSY AMDYWGQGTSVTVSS(SEQ ID No.:1)。
4. detection reagent as described in claim 1, the polyclonal antibody is prepared by the following method：
(a) CD19CAR scFv recombinant proteins are provided；With
(b) rabbit is immunized with the recombinant protein, to which acquisition is directed to the antiserum of the CD19CAR scFv recombinant proteins (i.e. Polyclonal antibody).
5. a kind of method preparing first antibody, which is characterized in that the first antibody is polyclonal antibody, and described more grams Grand antibody specificity is incorporated into the extracellular antigen binding domain of CD19CAR；
It the described method comprises the following steps：
(a) CD19CAR scFv recombinant proteins are provided；With
(b) rabbit is immunized with the recombinant protein, to which acquisition is directed to the antiserum of the CD19CAR scFv recombinant proteins (i.e. Polyclonal antibody).
6. a kind of CD19CAR detection kits, which is characterized in that the detection kit：
(t1) the first container, and the first antibody in the first container, the first antibody are polyclonal antibody, and And the polyclonal antibody specifically binds to the extracellular antigen binding domain of CD19CAR；With
(t2) second container, and positioned at the second container specificity be directed to the polyclonal antibody secondary antibody；With And
(t0) specification.
7. kit as claimed in claim 6, the secondary antibody carries detectable label.
8. kit as claimed in claim 7, the detection kit further includes containing one or more volumes selected from the group below Outer detection reagent：
(t3) third container, and the sealer in the third container；
(t4) the 4th container, and the positive control (such as CD19CAR scFv recombinant proteins) in the 4th container；With
(t5) the 5th container, and the negative control reagent in the 5th container.
9. the purposes of detection reagent as described in claim 1, which is characterized in that be used to prepare detection CD19CAR positive cells Reagent or kit.
10. a kind of method of detection CD19CAR positive cells, which is characterized in that including step：
(I) detection reagent described in claim 1 is provided；
(II) cell mass to be detected is detected with the detection reagent, to obtain determining for CD19CAR positive cells Property or quantitative testing result.
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